ABSTRACT: Studying essential or critical genes via knockout is exceptionally challenging due to associated lethality or inhibition of early developmental events that preclude temporal study of downstream processes. The Cre/Lox system has been a powerful tool to study these challenging genes but suffers from inefficiency and the need to customize reagents for each individual knockout. In order to overcome this issue in cultured cells, we previously developed a conditional rescue system to knockout essential genes by co-delivering an inducible cDNA rescue vector. A major barrier to the broad use of this approach is the low rate of gene modification and delivery of the conditional rescue cassette in some types of cells. These include hard to transfect cells, primary cells of various kinds, many stem/progenitor cells, and other cells that can't be cloned (i.e. propagated from single isolated cells). Fortuitously, recent advances using chemically modified gRNAs synthesized as RNA oligonucleotides combined with high quality Cas9 mRNA or protein has made it possible to gene edit primary human cells with unprecedented efficiencies. This initial work demonstrated that unmodified gRNAs were unable to induce detectable DSBs in primary human T or CD34+ cells, but the use of gRNAs synthesized as RNA oligonucleotides containing 3 tandem 2-O-methyl-3-phosphorothioate modified bases on the 5' and 3' ends could induce DSBs with efficiencies up to nearly 50% and 20%, respectively. We have utilized this system with an advanced electroporation platform (Neon) and achieved rates of gene editing >95% in primary human T or CD34+ hematopoietic stem cells (HSCs). More recently, a number of groups have utilized this technology combined with AAV (serotype 6) DNA donor delivery in human CD34+ HSCs and T cells for homologous recombination (HR) and attained HR frequencies as high as 43% and 60%, respectively. With these tools it is now conceivable to generate primary human lymphohematopoietic cells in a fashion similar to what has been done in genetically modified mouse models using the Cre/Lox for the last 30 years. Using chemically modified gRNAs combined with AAV donor delivery it is possible that simultaneous bi-allelic or sequential gene targeting could be used to engineer lymphohematopoietic cells with conditional, or conditional-temporal expression cassettes in a site-specific manner. Moreover, this approach combined with concurrent targeted gene knockout represents a modular and powerful system for studying basic biological questions using primary human lymphohematopoietic cells in a way never before possible. Thus, we propose to generate modular systems to knock-in conditional or conditional-temporal expression cassettes encoding a cDNA of a gene of interest in primary human T and CD34+ HSCs with concurrent knockout of the endogenous gene. If successful, this generic system could be used to study gene function with unprecedented control in primary human lymphohematopoietic cells in a way never previously possible, leading to new discoveries to improve human health.